RESUMO
Oncogene-induced STAT3-activation is central to tumor progression by promoting cancer cell expression of pro-angiogenic and immunosuppressive factors. STAT3 is also activated in infiltrating immune cells including tumor-associated macrophages (TAM) amplifying immune suppression. Consequently, STAT3 is considered as a target for cancer therapy. However, its interplay with other STAT-family members or transcription factors such as NF-κB has to be considered in light of their concerted regulation of immune-related genes. Here, we discuss new attempts at re-educating immune suppressive tumor-associated macrophages towards a CD8 T cell supporting profile, with an emphasis on the role of STAT transcription factors on TAM functional programs. Recent clinical trials using JAK/STAT inhibitors highlighted the negative effects of these molecules on the maintenance and function of effector/memory T cells. Concerted regulation of STAT3 and STAT5 activation in CD8 T effector and memory cells has been shown to impact their tumor-specific responses including intra-tumor accumulation, long-term survival, cytotoxic activity and resistance toward tumor-derived immune suppression. Interestingly, as an escape mechanism, melanoma cells were reported to impede STAT5 nuclear translocation in both CD8 T cells and NK cells. Ours and others results will be discussed in the perspective of new developments in engineered T cell-based adoptive therapies to treat cancer patients.
RESUMO
Despite impressive clinical success, cancer immunotherapy based on immune checkpoint blockade remains ineffective in many patients due to tumoral resistance. Here we use the autochthonous TiRP melanoma model, which recapitulates the tumoral resistance signature observed in human melanomas. TiRP tumors resist immunotherapy based on checkpoint blockade, cancer vaccines or adoptive T-cell therapy. TiRP tumors recruit and activate tumor-specific CD8+ T cells, but these cells then undergo apoptosis. This does not occur with isogenic transplanted tumors, which are rejected after adoptive T-cell therapy. Apoptosis of tumor-infiltrating lymphocytes can be prevented by interrupting the Fas/Fas-ligand axis, and is triggered by polymorphonuclear-myeloid-derived suppressor cells, which express high levels of Fas-ligand and are enriched in TiRP tumors. Blocking Fas-ligand increases the anti-tumor efficacy of adoptive T-cell therapy in TiRP tumors, and increases the efficacy of checkpoint blockade in transplanted tumors. Therefore, tumor-infiltrating lymphocytes apoptosis is a relevant mechanism of immunotherapy resistance, which could be blocked by interfering with the Fas/Fas-ligand pathway.
Assuntos
Imunoterapia , Linfócitos do Interstício Tumoral/imunologia , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Animais , Apoptose/imunologia , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Proteína Ligante Fas/antagonistas & inibidores , Proteína Ligante Fas/genética , Proteína Ligante Fas/imunologia , Feminino , Humanos , Imunoterapia/métodos , Imunoterapia Adotiva , Linfócitos do Interstício Tumoral/patologia , Masculino , Melanoma Experimental/patologia , Camundongos , Camundongos Transgênicos , Microambiente Tumoral/imunologia , Receptor fas/antagonistas & inibidores , Receptor fas/imunologiaRESUMO
Cancer-germline genes in both humans and mice have been shown to encode antigens susceptible to targeting by cytotoxic CD8 T effector cells (CTL). We analysed the ability of CTL to kill different tumour cell lines expressing the same cancer-germline gene P1A (Trap1a). We previously demonstrated that CTL expressing a T-cell receptor specific for the P1A35-43 peptide associated with H-2Ld , although able to induce regression of P1A-expressing P815 mastocytoma cells, were much less effective against P1A-expressing melanoma cells. Here, we analysed parameters of the in vitro interaction between P1A-specific CTL and mastocytoma or melanoma cells expressing similar levels of the P1A gene and of surface H-2Ld . The mastocytoma cells were more sensitive to cytolysis than the melanoma cells in vitro. Analysis by video-microscopy of early events required for target cell killing showed that similar patterns of increase in cytoplasmic Ca2+ concentration ([Ca2+ ]i) were induced by both types of P1A-expressing tumour cells. However, the use of CTL expressing a fluorescent granzyme B (GZMB-Tom) showed a delay in the migration of cytotoxic granules to the tumour interaction site, as well as a partially deficient GZMB-Tom exocytosis in response to the melanoma cells. Among surface molecules possibly affecting tumour-CTL interactions, the mastocytoma cells were found to express intercellular adhesion molecule-1, the ligand for LFA-1, which was not detected on the melanoma cells.
Assuntos
Antígenos de Neoplasias/metabolismo , Exocitose , Mastocitoma/imunologia , Melanoma/imunologia , Fragmentos de Peptídeos/metabolismo , Vesículas Secretórias/metabolismo , Linfócitos T Citotóxicos/imunologia , Animais , Antígenos de Neoplasias/genética , Sinalização do Cálcio , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Antígeno de Histocompatibilidade H-2D/metabolismo , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Ativação Linfocitária , Antígeno-1 Associado à Função Linfocitária/metabolismo , Camundongos , Especificidade do Receptor de Antígeno de Linfócitos TRESUMO
T cells infiltrating neoplasms express surface molecules typical of chronically virus-stimulated T cells, often termed "exhausted" T cells. We compared the transcriptome of "exhausted" CD8 T cells infiltrating autochthonous melanomas to those of naïve and acutely stimulated CD8 T cells. Despite strong similarities between transcriptional signatures of tumor- and virus-induced exhausted CD8 T cells, notable differences appeared. Among transcriptional regulators, Nr4a2 and Maf were highly overexpressed in tumor-exhausted T cells and significantly upregulated in CD8 T cells from human melanoma metastases. Transduction of murine tumor-specific CD8 T cells to express Maf partially reproduced the transcriptional program associated with tumor-induced exhaustion. Upon adoptive transfer, the transduced cells showed normal homeostasis but failed to accumulate in tumor-bearing hosts and developed defective anti-tumor effector responses. We further identified TGFß and IL-6 as main inducers of Maf expression in CD8 T cells and showed that Maf-deleted tumor-specific CD8 T cells were much more potent to restrain tumor growth in vivo. Therefore, the melanoma microenvironment contributes to skewing of CD8 T cell differentiation programs, in part by TGFß/IL-6-mediated induction of Maf.
Assuntos
Linfócitos T CD8-Positivos/citologia , Diferenciação Celular/imunologia , Melanoma/metabolismo , Proteínas Proto-Oncogênicas c-maf/metabolismo , Microambiente Tumoral/fisiologia , Animais , Linfócitos T CD8-Positivos/metabolismo , Primers do DNA/genética , Citometria de Fluxo , Perfilação da Expressão Gênica , Proteínas de Homeodomínio/genética , Interleucina-6/metabolismo , Luciferases , Camundongos , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-maf/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Crescimento Transformador beta/metabolismoRESUMO
Tumors with reduced expression of MHC class I (MHC-I) molecules may be unrecognized by tumor antigen-specific CD8+ T cells and thus constitute a challenge for cancer immunotherapy. Here we monitored development of autochthonous melanomas in TiRP mice that develop tumors expressing a known tumor antigen as well as a red fluorescent protein (RFP) reporter knock in gene. The latter permits non-invasive monitoring of tumor growth by biofluorescence. One developing melanoma was deficient in cell surface expression of MHC-I, but MHC-I expression could be rescued by exposure of these cells to IFNγ. We show that CD8+ T cells specific for tumor antigen/MHC-I were efficient at inducing regression of the MHC-I-deficient melanoma, provided that the T cells were endowed with properties permitting their migration into the tumor and their efficient production of IFNγ. This was the case for CD8+ T cells transfected to express an active form of STAT5 (STAT5CA). The amount of IFNγ produced ex vivo from T cells present in tumors after adoptive transfer of the CD8+ T cells was correlated with an increase in surface expression of MHC-I molecules by the tumor cells. We also show that these CD8+ T cells expressed PD-1 and upregulated its ligand PDL-1 on melanoma cells within the tumor. Despite upregulation of this immunosuppressive pathway, efficient IFNγ production in the melanoma microenvironment was found associated with resistance of STAT5CA-expressing CD8+ T cells to inhibition both by PD-1/PDL-1 engagement and by TGFß1, two main immune regulatory mechanisms hampering the efficiency of immunotherapy in patients.
RESUMO
CD8 T cells used in adoptive immunotherapy may be manipulated to optimize their effector functions, tissue-migratory properties and long-term replicative potential. We reported that antigen-stimulated CD8 T cells transduced to express an active form of the transcription factor signal transducer and activator of transcription 5 (STAT5CA) maintained these properties upon adoptive transfer. We now report on the requirements of STAT5CA-expressing CD8 T cells for cell survival and proliferation in vivo. We show that STAT5CA expression allows for greater expansion of T cells in vivo, while preserving dependency on T-cell-receptor-mediated tonic stimulation for their in vivo maintenance and return to a quiescent stage. STAT5CA expression promotes the formation of a large pool of effector memory T cells that respond upon re-exposure to antigen and present an increased sensitivity to γc receptor cytokine engagement for STAT5 phosphorylation. In addition, STAT5CA expression prolongs the survival of what would otherwise be short-lived terminally differentiated KLRG1-positive effector cells with up-regulated expression of the senescence-associated p16(INK) (4A) transcripts. However, development of a KLRG1-positive CD8 T cell population was independent of either p16(INK) (4A) or p19(ARF) expression (as shown using T cells from CDKN2A(-/-) mice) but was associated with expression of transcripts encoding p15(INK) (4B) , another protein involved in senescence induction. We conclude that T-cell-receptor- and cytokine-dependent regulation of effector T cell homeostasis, as well as mechanisms leading to senescent features of a population of CD8 T cells are maintained in STAT5CA-expressing CD8 T cells, even for cells that are genetically deficient in expression of the tumour suppressors p16(INK) (4A) and p19(ARF) .
Assuntos
Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular/imunologia , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p15/imunologia , Inibidor p16 de Quinase Dependente de Ciclina/imunologia , Fator de Transcrição STAT5/imunologia , Animais , Diferenciação Celular/genética , Senescência Celular/genética , Senescência Celular/imunologia , Inibidor de Quinase Dependente de Ciclina p15/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Regulação da Expressão Gênica/imunologia , Lectinas Tipo C , Camundongos , Camundongos Knockout , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia , Fator de Transcrição STAT5/genéticaRESUMO
Several recent studies have implicated myeloid cells in providing a microenvironment that promotes tumor cell survival and metastasis, therefore preparing a "premetastatic niche" for cancer progression. In this issue of the European Journal of Immunology, Zhang et al. [Eur. J. Immunol. 2015. 45: 71-81] address the regulation of immune cells in premetastatic lymph nodes in experimental mouse models. The authors show that signal transducer and activator of transcription 3 (STAT3) ablation in murine myeloid cells, which renders the premetastatic niche less receptive to metastasis by B16 melanoma cells, also leads to local activation in the niche of CD8(+) T cells with increased expression of IFN-γ and granzyme B. Data further suggest that STAT3 activation in the myeloid population leads to poor tumor antigen presenting capacity as well as resistance to CD8(+) T-cell killing. Based on these studies in mice and observations in human cancer patients, the authors propose treatments designed to regulate STAT3 activation, which are correlated with increased cytolytic activity of CD8(+) T cells in mouse models.
Assuntos
Regulação Neoplásica da Expressão Gênica/imunologia , Vigilância Imunológica , Macrófagos Peritoneais/imunologia , Melanoma Experimental/imunologia , Neoplasias Cutâneas/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , HumanosRESUMO
CD8 T cells contribute to long-term protection against Listeria monocytogenes infection by differentiating into memory T cells. These rapidly respond to antigen or inflammation upon secondary infection. In this study we used CD8 T cells from OT1 mice and CD4 T cells from OT2 mice expressing a fluorescent chimeric granzyme (GZMB-Tom) protein to monitor the primary response to infection with ovalbumin-expressing L. monocytogenes (Lm-OVA). We show that, unlike poorly responding CD4 T cells, CD8 T cells readily proliferated and expressed high levels of GZMB-Tom as early as 2 days after infection. FACS analysis showed GZMB-Tom expression in undivided CD8 T cells, with its level increasing over one to four divisions. OT1 T cells were visualized in the T-cell zone by confocal microscopy. This showed GZMB-Tom-containing granules oriented towards MHCII-positive cells. Twenty hours later, most OT1 T cells had divided but their level of GZMB-Tom expression was reduced. Recently divided OT1 cells failed to express GZMB-Tom. Fourteen hours after secondary infection, GZMB-Tom was re-expressed in memory OT1 T cells responding either to Lm-OVA or L. monocytogenes. Differences in the activation phenotype and in the splenic distribution of OT1 T cells were observed, depending on the challenge. Notably, OTI T cells with polarized granules were only observed after challenge with cognate antigen. This work showed that the GZMB-Tom knock-in mice in which GZMB-Tom faithfully reproduced GZMB expression, provide useful tools to dissect mechanisms leading to the development of anti-bacterial effector and memory CD8 T cells and reactivation of the memory response to cognate antigen or inflammatory signals.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Proliferação de Células , Regulação Enzimológica da Expressão Gênica/imunologia , Granzimas/imunologia , Listeria monocytogenes/imunologia , Listeriose/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/patologia , Regulação Enzimológica da Expressão Gênica/genética , Granzimas/genética , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Listeriose/genética , Listeriose/patologia , Camundongos , Camundongos Transgênicos , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Fatores de TempoRESUMO
The transcription factor NF-κB is central to inflammatory signaling and activation of innate and adaptive immune responses. Activation of the NF-κB pathway is tightly controlled by several negative feedback mechanisms, including A20, an ubiquitin-modifying enzyme encoded by the tnfaip3 gene. Mice with selective deletion of A20 in myeloid, dendritic, or B cells recapitulate some human inflammatory pathology. As we observed high expression of A20 transcripts in dysfunctional CD8 T cells in an autochthonous melanoma, we analyzed the role of A20 in regulation of CD8 T-cell functions, using mice in which A20 was selectively deleted in mature conventional T cells. These mice developed lymphadenopathy and some organ infiltration by T cells but no splenomegaly and no detectable pathology. A20-deleted CD8 T cells had increased sensitivity to antigen stimulation with production of large amounts of IL-2 and IFNγ, correlated with sustained nuclear expression of NF-κB components reticuloendotheliosis oncogene c-Rel and p65. Overexpression of A20 by retroviral transduction of CD8 T cells dampened their intratumor accumulation and antitumor activity. In contrast, relief from the A20 brake in NF-κB activation in adoptively transferred antitumor CD8 T cells led to improved control of melanoma growth. Tumor-infiltrating A20-deleted CD8 T cells had enhanced production of IFNγ and TNFα and reduced expression of the inhibitory receptor programmed cell death 1. As manipulation of A20 expression in CD8 T cells did not result in pathologic manifestations in the mice, we propose it as a candidate to be targeted to increase antitumor efficiency of adoptive T-cell immunotherapy.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Cisteína Endopeptidases/imunologia , Imunidade Celular , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Melanoma/imunologia , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/patologia , Cisteína Endopeptidases/genética , Humanos , Interferon gama/genética , Interferon gama/imunologia , Interleucina-2/genética , Interleucina-2/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Melanoma/genética , Melanoma/patologia , Camundongos , Camundongos Knockout , NF-kappa B/genética , NF-kappa B/imunologia , Proteína 3 Induzida por Fator de Necrose Tumoral alfa , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Mechanisms controlling immune reactivity prevent excessive inflammation and autoimmunity, but generally dampen antitumor activity. We recently showed that adoptively transferred antitumor CD8+ T cells harboring a deletion of A20/Tnfaip3, a molecule controlling NF-κB activation, possessed heightened antitumor activity in vivo. The boosted immunity of A20-deleted CD8+ T cells correlated with a heightened capacity to produce IFNγ and TNFα while expressing reduced levels of the immune checkpoint molecule PD-1.
RESUMO
In adoptive therapy, CD8 T cells expressing active STAT5 (STAT5CA) transcription factors were found to be superior to unmanipulated counterparts in long-term persistence, capacity to infiltrate autochthonous mouse melanomas, thrive in their microenvironment, and induce their regression. However, the molecular mechanisms sustaining these properties were undefined. In this study, we report that STAT5CA induced sustained expression of genes controlling tissue homing, cytolytic granule composition, type 1 CD8 cytotoxic T cell-associated effector molecules granzyme B(+), IFN-γ(+), TNF-α(+), and CCL3(+), but not IL-2, and transcription factors T-bet and eomesodermin (Eomes). Chromatin immunoprecipitation sequencing analyses identified the genes possessing regulatory regions to which STAT5 bound in long-term in vivo maintained STAT5CA-expressing CD8 T cells. This analysis identified 34% of the genes differentially expressed between STAT5CA-expressing and nonexpressing effector T cells as direct STAT5CA target genes, including those encoding T-bet, Eomes, and granzyme B. Additionally, genes encoding the IL-6R and TGFbRII subunits were stably repressed, resulting in dampened IL-17-producing CD8 T cell polarization in response to IL-6 and TGF-ß1. The absence of T-bet did not affect STAT5CA-driven accumulation of the T cells in tissue or their granzyme B expression but restored IL-2 secretion and IL-6R and TGFbRII expression and signaling, as illustrated by IL-17 induction. Therefore, concerted STAT5/T-bet/Eomes regulation controls homing, long-term maintenance, recall responses, and resistance to polarization towards IL-17-producing CD8 T cells while maintaining expression of an efficient type 1 CD8 cytotoxic T cell program (granzyme B(+), IFN-γ(+)).
Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Interleucina-6/metabolismo , Fator de Transcrição STAT5/metabolismo , Proteínas com Domínio T/genética , Fator de Crescimento Transformador beta1/metabolismo , Animais , Linfócitos T CD8-Positivos/citologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Interleucina-17/metabolismo , Camundongos , Camundongos Transgênicos , Receptores de Citocinas/genética , Receptores de Citocinas/metabolismo , Fator de Transcrição STAT5/genética , Transdução de Sinais , Proteínas com Domínio T/deficiência , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Transcrição GênicaRESUMO
To evaluate acquisition and activation of cytolytic functions during immune responses we generated knock in (KI) mice expressing Granzyme B (GZMB) as a fusion protein with red fluorescent tdTomato (GZMB-Tom). As for GZMB in wild type (WT) lymphocytes, GZMB-Tom was absent from naïve CD8 and CD4 T cells in GZMB-Tom-KI mice. It was rapidly induced in most CD8 T cells and in a subpopulation of CD4 T cells in response to stimulation with antibodies to CD3/CD28. A fraction of splenic NK cells expressed GZMB-Tom ex vivo with most becoming positive upon culture in IL-2. GZMB-Tom was present in CTL granules and active as a protease when these degranulated into cognate target cells, as shown with target cells expressing a specific FRET reporter construct. Using T cells from mice expressing GZMB-Tom but lacking perforin, we show that the transfer of fluorescent GZMB-Tom into target cells was dependent on perforin, favoring a role for perforin in delivery of GZMB at the target cells' plasma membranes. Time-lapse video microscopy showed Ca++ signaling in CTL upon interaction with cognate targets, followed by relocalization of GZMB-Tom-containing granules to the synaptic contact zone. A perforin-dependent step was next visualized by the fluorescence signal from the non-permeant dye TO-PRO-3 at the synaptic cleft, minutes before the labeling of the target cell nucleus, characterizing a previously undescribed synaptic event in CTL cytolysis. Transferred OVA-specific GZMB-Tom-expressing CD8 T cells acquired GZMB-Tom expression in Listeria monocytogenes-OVA infected mice as soon as 48h after infection. These GZMB-Tom positive CD8 T cells localized in the splenic T-zone where they interacted with CD11c positive dendritic cells (DC), as shown by GZMB-Tom granule redistribution to the T/DC contact zone. GZMB-Tom-KI mice thus also provide tools to visualize acquisition and activation of cytolytic function in vivo.
Assuntos
Degranulação Celular , Granzimas/biossíntese , Vesículas Secretórias/metabolismo , Linfócitos T Citotóxicos/fisiologia , Animais , Diferenciação Celular , Células Cultivadas , Expressão Gênica , Técnicas de Introdução de Genes , Granzimas/genética , Células Matadoras Naturais/metabolismo , Cinética , Listeriose/imunologia , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/genética , Linfonodos/imunologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Confocal , Microscopia de Fluorescência , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Vesículas Secretórias/enzimologia , Análise de Célula Única , Proteína Vermelha FluorescenteRESUMO
Discovery of tumor antigen (TA) recognized by autologous T cells (TCs) in patients with melanoma has led to clinical protocols using either vaccination or adoptive transfer of TA-specific TCs. However, efficacy of these treatments has been hampered by inhibitory effects exerted on tumor-infiltrating TCs by tumor-intrinsic mediators or by recruitment of immunosuppressive cells. A mouse model of autochthonous melanoma recapitulates some aspects of inflammatory melanoma development in patients. These include a systemic Th2-/Th17-oriented chronic inflammation, recruitment of immunosuppressive myeloid cells and acquisition by tumor-infiltrating TCs of an 'exhausted' phenotype characterized by expression of multiple inhibitory receptors including programmed death-1, also expressed on patients' melanoma-infiltrating TCs. Rather than using extracellular blocking reagents to inhibitory surface molecules on TCs, we sought to dampen negative signaling exerted on them. Adoptively transferred TCs presenting increased cytokine receptor signaling due to expression of an active Stat5 transcription factor were efficient at inducing melanoma regression in the preclinical melanoma model. These transferred TCs thrived and retained expression of effector molecules in the melanoma microenvironment, defining a protocol endowing TCs with the ability to resist melanoma-induced immunosuppression.
Assuntos
Transferência Adotiva , Terapia de Imunossupressão , Inflamação/patologia , Melanoma/imunologia , Melanoma/terapia , Linfócitos T/imunologia , Linfócitos T/patologia , Imunidade Adaptativa/imunologia , Animais , Antígenos de Neoplasias/imunologia , Humanos , Imunoterapia Adotiva , Linfócitos do Interstício Tumoral/metabolismo , Melanoma/patologia , Camundongos , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/terapiaRESUMO
We compared gene expression signatures of aggressive amelanotic (Amela) melanomas with those of slowly growing pigmented melanomas (Mela), identifying pathways potentially responsible for the aggressive Amela phenotype. Both tumors develop in mice upon conditional deletion in melanocytes of Ink4a/Arf tumor suppressor genes with concomitant expression of oncogene H-Ras(G12V) and a known tumor antigen. We previously showed that only the aggressive Amela tumors were highly infiltrated by leukocytes concomitant with local and systemic inflammation. We report that Amela tumors present a pattern of de-differentiation with reduced expression of genes involved in pigmentation. This correlates with reduced and enhanced expression, respectively, of microphthalmia-associated (Mitf) and Pou3f2/Brn-2 transcription factors. The reduced expression of Mitf-controlled melanocyte differentiation antigens also observed in some human cutaneous melanoma has important implications for immunotherapy protocols that generally target such antigens. Induced Amela tumors also express Epithelial-Mesenchymal-Transition (EMT)-like and TGFß-pathway signatures. These are correlated with constitutive Smad3 signaling in Amela tumors and melanoma cell lines. Signatures of infiltrating leukocytes and some chemokines such as chemotactic cytokine ligand 2 (Ccl2) that contribute to leukocyte recruitment further characterize Amela tumors. Inhibition of the mitogen-activated protein kinase (MAPK) activation pathway in Amela tumor lines leads to reduced expression of EMT hallmark genes and inhibits both proinflammatory cytokine Ccl2 gene expression and Ccl2 production by the melanoma cells. These results indicate a link between EMT-like processes and alterations of immune functions, both being controlled by the MAPK pathway. They further suggest that targeting the MAPK pathway within tumor cells will impact tumor-intrinsic oncogenic properties as well as the nature of the tumor microenvironment.
Assuntos
Transição Epitelial-Mesenquimal , Melanoma Amelanótico/patologia , Transdução de Sinais , Neoplasias Cutâneas/patologia , Fator de Crescimento Transformador beta/metabolismo , Animais , Diferenciação Celular , Linhagem Celular Tumoral , Quimiocina CCL2/metabolismo , Regulação para Baixo , Ativação Enzimática , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Sistema de Sinalização das MAP Quinases , Melanócitos/metabolismo , Melanócitos/patologia , Melanoma Amelanótico/genética , Melanoma Amelanótico/metabolismo , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas do Tecido Nervoso/genética , Fatores do Domínio POU/genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Proteína Smad3/metabolismo , Regulação para CimaRESUMO
Central tolerance toward tissue-restricted Ags is considered to rely on ectopic expression in the thymus, which was also observed for tumor Ags encoded by cancer-germline genes. It is unknown whether endogenous expression shapes the T cell repertoire against the latter Ags and explains their weak immunogenicity. We addressed this question using mouse cancer-germline gene P1A, which encodes antigenic peptide P1A(35-43) presented by H-2L(d). We made P1A-knockout (P1A-KO) mice and asked whether their anti-P1A(35-43) immune responses were stronger than those of wild-type mice and whether P1A-KO mice responded to other P1A epitopes, against which wild-type mice were tolerized. We observed that both types of mice mounted similar P1A(35-43)-specific CD8 T cell responses, although the frequency of P1A(35-43)-specific CD8 T cells generated in response to P1A-expressing tumors was slightly higher in P1A-KO mice. This higher reactivity allowed naive P1A-KO mice to reject spontaneously P1A-expressing tumors, which progressed in wild-type mice. TCR-Vß usage of P1A(35-43)-specific CD8 cells was slightly modified in P1A-KO mice. Peptide P1A(35-43) remained the only P1A epitope recognized by CD8 T cells in both types of mice, which also displayed similar thymic selection of a transgenic TCR recognizing P1A(35-43). These results indicate the existence of a minimal tolerance to an Ag encoded by a cancer-germline gene and suggest that its endogenous expression only slightly affects diversification of the T cell repertoire against this Ag.
Assuntos
Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/imunologia , Epitopos/imunologia , Tolerância Imunológica , Neoplasias/imunologia , Peptídeos/imunologia , Animais , Antígenos de Neoplasias/genética , Linhagem Celular Tumoral , Epitopos/genética , Camundongos , Camundongos Knockout , Neoplasias/genética , Peptídeos/genética , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/imunologiaRESUMO
Immunotherapy based on adoptive transfer of tumor antigen-specific CD8(+) T cell (TC) is generally limited by poor in vivo expansion and tumor infiltration. In this study, we report that activated STAT5 transcription factors (STAT5CA) confer high efficiency on CD8(+) effector T cells (eTC) for host colonization after adoptive transfer. Engineered expression of STAT5CA in antigen-experienced TCs with poor replicative potential was also sufficient to convert them into long-lived antigen-responsive eTCs. In transplanted mastocytoma- or melanoma-bearing hosts, STAT5CA greatly enhanced the ability of eTCs to accumulate in tumors, become activated by tumor antigens, and to express the cytolytic factor granzyme B. Taken together, these properties contributed to an increase in tumor regression by STAT5CA-transduced, as compared with untransduced, TCs including when the latter control cells were combined with infusion of interleukin (IL)-2/anti-IL-2 complexes. In tumors arising in the autochthonous TiRP transgenic model of melanoma associated with systemic chronic inflammation, endogenous CD8(+) TCs were nonfunctional. In this setting, adoptive transfer of STAT5CA-transduced TCs produced superior antitumor effects compared with nontransduced TCs. Our findings imply that STAT5CA expression can render TCs resistant to the immunosuppressive environment of melanoma tumors, enhancing their ability to home to tumors and to maintain high granzyme B expression, as well as their capacity to stimulate granzyme B expression in endogenous TCs.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Melanoma/fisiopatologia , Fator de Transcrição STAT5/fisiologia , Transferência Adotiva , Animais , Citometria de Fluxo , Melanoma/patologia , Melanoma/terapia , Camundongos , Transplante HeterólogoRESUMO
Migration of dendritic cells (DC) from the tumor environment to the T cell cortex in tumor-draining lymph nodes (TDLN) is essential for priming naïve T lymphocytes (TL) to tumor antigen (Ag). We used a mouse model of induced melanoma in which similar oncogenic events generate two phenotypically distinct melanomas to study the influence of tumor-associated inflammation on secondary lymphoid organ (SLO) organization. One tumor promotes inflammatory cytokines, leading to mobilization of immature myeloid cells (iMC) to the tumor and SLO; the other does not. We report that inflammatory tumors induced alterations of the stromal cell network of SLO, profoundly altering the distribution of TL and the capacity of skin-derived DC and TL to migrate or home to TDLN. These defects, which did not require tumor invasion, correlated with loss of fibroblastic reticular cells in T cell zones and in impaired production of CCL21. Infiltrating iMC accumulated in the TDLN medulla and the splenic red pulp. We propose that impaired function of the stromal cell network during chronic inflammation induced by some tumors renders spleens non-receptive to TL and TDLN non-receptive to TL and migratory DC, while the entry of iMC into these perturbed SLO is enhanced. This could constitute a mechanism by which inflammatory tumors escape immune control. If our results apply to inflammatory tumors in general, the demonstration that SLO are poorly receptive to CCR7-dependent migration of skin-derived DC and naïve TL may constitute an obstacle for proposed vaccination or adoptive TL therapies of their hosts.
Assuntos
Movimento Celular/imunologia , Células Dendríticas/imunologia , Inflamação/patologia , Linfonodos/patologia , Melanoma/patologia , Baço/patologia , Linfócitos T/imunologia , Animais , Linfócitos B/imunologia , Antígeno CD11b/metabolismo , Diferenciação Celular/imunologia , Quimiocina CCL21/metabolismo , Células Dendríticas/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Inflamação/complicações , Inflamação/imunologia , Linfonodos/imunologia , Melanoma/complicações , Melanoma/imunologia , Glicoproteínas de Membrana/metabolismo , Camundongos , Células Mieloides/imunologia , Células Mieloides/patologia , Pele/imunologia , Pele/patologia , Neoplasias Cutâneas/complicações , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/patologia , Baço/imunologia , Células Estromais/imunologia , Células Estromais/patologia , Linfócitos T/patologiaRESUMO
Peptide/MHC complexes recognized by alloreactive T lymphocytes (TLs) have been identified, but their contribution to in vivo allo-rejection is not known. We previously characterized the peptide pBM1, highly represented among endogenous H-2K(b) (K(b) )-associated peptides and critically required to induce full activation of H-2(k) monoclonal CD8(+) TLs expressing the cognate TCR-BM3.3. Here, we asked whether a pBM1/K(b) -specific TL subset could be detected within a polyclonal TL population rejecting allogeneic cells in vivo. We show that the proportion of pBM1/K(b) -binding CD8(+) TLs increased from <0.04% in naïve mice to 3% of activated CD44(+) CD8(+) TLs in H-2(k) mice rejecting K(b) -expressing cells. Among these, TCR-Vß2 usage was greatly enriched, and 75% of them shared a TCR-Vß2 CDR3ß motif with the prototype TCR-BM3.3. Fewer than 5% of K(b) -reactive CD44(+) CD8(+) TLs not binding pBM1/K(b) displayed this CDR3ß motif. We found that the recurrent CDR3ß motif of pBM1/K(b) -binding TLs was assembled from distinct V/D/J recombination events, suggesting that it is recruited upon immunization for its optimal TCR-peptide/MHC fit. Thus, a CDR3ß motif generated by a process akin to "convergent recombination" accounts for a sizable fraction of the alloreactive anti-K(b) TCR repertoire.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Regiões Determinantes de Complementaridade/imunologia , Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Motivos de Aminoácidos/imunologia , Sequência de Aminoácidos , Animais , Linfócitos T CD8-Positivos/metabolismo , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/metabolismo , Citometria de Fluxo , Antígenos H-2/imunologia , Antígenos H-2/metabolismo , Receptores de Hialuronatos/imunologia , Receptores de Hialuronatos/metabolismo , Imunização , Camundongos , Camundongos Endogâmicos CBA , Peptídeos/metabolismo , Ligação Proteica/imunologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Recombinação Genética/imunologia , Análise de Sequência de DNA , Éxons VDJ/genética , Éxons VDJ/imunologiaRESUMO
We studied the effect of the immune system on two differentially aggressive melanomas developing in mice on conditional deletion of the INK4A/ARF tumor suppressor gene, with concomitant expression of oncogene H-Ras(G12V) and a natural cancer-germline tumor antigen (TA). "Slow progressor" melanomas contained no activated T lymphocytes (TL). In contrast, "aggressive" melanomas were infiltrated by activated TLs lacking effector molecules and expressing high levels of PD-1, indicating an exhausted phenotype. Aggressive melanomas were also infiltrated by immature myeloid cells (IMC). Infiltration was associated with local inflammation and systemic Th2/Th17-oriented chronic inflammation that seemed to impair further activation of TLs, as tumor-specific T cells adoptively transferred into mice bearing aggressive melanomas were poorly activated and failed to infiltrate the melanoma. This immunosuppression also led to the incapacity of these mice to reject inoculated TA-positive tumors, in contrast to slow-progressing melanoma-bearing mice, which were responsive. To test the role of adaptive immunity in tumor progression, we induced melanomas in immunodeficient RagKO compound mice. These mice developed aggressive but not slow-progressing melanomas at a higher frequency and with a shorter latency than immunocompetent mice. Immunodeficient mice also developed abnormal inflammation and infiltration of IMCs in a manner similar to immunocompetent mice, indicating that this phenotype was not dependent on adaptive immunity. Therefore, tumor-intrinsic factors distinguishing the two melanoma types control the initiation of inflammation, which was independent of adaptive immunity. The latter delayed development of aggressive melanomas but was overridden by inflammation.